Convergent Synthesis for Kahalalide Compounds

ABSTRACT

New synthetic routes to kahalalide compounds are provided. These are based on convergent approaches using orthogonal protecting schemes, where a better control of the intermediates is taken. Kahalalide F or a mimic of kahalalide F is synthesised by coupling a cyclic part with a side chain fragment, e.g. according to the reaction (1)

FIELD OF THE INVENTION

The present invention is directed to new synthetic routes for kahalalidecompounds and related compounds.

BACKGROUND OF THE INVENTION

The kahalalide compounds are peptides isolated from a Hawaiianherbivorous marine species of mollusc, Elysia rufescens and its diet,the green alga Bryopsis sp. Kahalalide F is described in Hamann et al.,J. Am. Chem. Soc., 1993, 115, 5825-5826.

Kahalalide A-G are described in Hamann, M. et al., J. Org. Chem, 1996,61, 6594-6600: “Kahalalides: bioactive peptides from a marine molluskElysia rufescens and its algal diet Bryopsis sp.” Kahalalide F has thefollowing structure:

Kahalalide H and J are described in Scheuer P. J. et al., J. Nat. Prod.1997, 60, 562-567: “Two acyclic kahalalides from the sacoglossan molluskElysia nufescens”.

Kahalalide O is described in Scheuer P. J. et al., J. Nat. Prod. 2000,63(1) 152-4: “A new depsipeptide from the sacoglossan mollusk Elysiaornata and the green alga Bryopsis species”.

For kahalalide K, see Kan, Y. et al., J. Nat. Prod. 1999 62(8) 1169-72:“Kahalalide K: A new cyclic depsipeptide from the Hawaiian green algabryopsis species”.

For related reports, see also Goetz et al., Tetrahedron, 1999, 55;7739-7746: “The absolute stereochemistry of Kahalalide F”; Albericio, F.et al. Tetrahedron Letters, 2000, 41, 9765-9769: “Kahalalide B.Synthesis of a natural cyclodepsipeptide”; Becerro et al. J. Chem. Ecol.2001, 27(11), 2287-99: “Chemical defenses of the sarcoglossan molluskElysia rufescens and its host Alga bryopsis sp.”.

Of the kahalalide compounds, kahalalide F and analogues (Formula 1below) are the most promising because of antitumoral activities. Thestructure is complex, comprising six amino acids as a cyclic part, andan exocyclic chain of seven amino acids with a terminal aliphatic/fattyacid group.

Among those of more interesting activity is when R is a 5-methylhexanoylor the isomer where R is a 4(S)-methylhexanoyl group.

The activity of kahalalide F against in vitro cell cultures of humanlung carcinoma A-549 and human colon carcinoma HT-29 were reported in EP610 078. Kahalalide F has also demonstrated to have antiviral andantifungal properties, as well as to be useful in the treatment ofpsoriasis.

WO 02 36145 describes pharmaceutical compositions containing kahalalideF and new uses of this compound in cancer therapy and is incorporatedherein by reference its entirety.

WO 03 33012 describes the clinical use in oncology of kahalalidecompounds and is incorporated herein by specific reference in itsentirety.

The synthesis and cytotoxic activities of natural and synthetickahalalide compounds is described in WO 01 58934, which is incorporatedherein by specific reference in its entirety. WO 01 58934 describes thesynthesis of kahalalide F and also of mimic compounds with a similarstructure in which amino acids are replaced by other amino acids or theterminal fatty acid chain is replaced by other fatty acids. Inparticular, it describes a solid phase synthesis of kahalalide F (I) andderivatives and analogues, in accordance with the following scheme:

where the various groups take the meanings given in WO 01 58934. Solidphase synthesis is employed to generate a partially protected open chaincompound, which is then cleaved, cyclized and deprotected.

WO 2004035613 relates to the 4(S)-methylhexanoyl isomer mentioned aboveand other compounds.

WO 2005023846 describes the synthesis of more mimic compounds with asimilar structure of kahalalide F in which amino acids are replaced byother amino acids or the terminal fatty acid chain is replaced by otheraliphatic/fatty acids. It uses the synthetic route of WO 01 58934. WO2005023846 is incorporated herein by specific reference in its entirety.

There is still a need to provide synthetic routes for kahalalidecompounds.

SUMMARY OF THE INVENTION

We have found several new improved routes for the preparation ofkahalalide analogues. The previous strategy was based in a stepwisesolid-phase synthesis of the partial protected open chain of thekahalalide, followed by the cyclization carried out in solution, andfinally, a removal of the protecting groups in solution as well.

The new routes are based in convergent approaches, where a bettercontrol of the intermediates is taken, with more reactions carried outin solution, and therefore with more characterization of theintermediates.

The invention is also directed to a process for the preparation of newanalogues of parent compounds.

Thus, the present invention provides a synthetic route to naturalkahalalides, especially kahalalide F, and mimics of natural kahalalides.The mimic compounds may differ in one or more amino acids, and/or one ormore components of the acyl side chain.

Suitably the mimics of this invention have at least one of the followingfeatures to differentiate from a parent naturally occurring kahalalide:

1 to 7, especially 1 to 3, more especially 1 or 2, most especially 1,amino acid which is not the same as an amino acid of the parentcompound;

1 to 10, especially 1 to 6, more especially 1 to 3, most especially 1 or2, additional methylene groups in the side chain acyl group of theparent compound;

1 to 10, especially 1 to 6, more especially 1 to 3, most especially 1 pr2, methylene groups omitted from the side chain acyl group of the parentcompound;

1 to 6, especially 1 to 3, more especially 1 or 3, substituents added toor omitted from the side chain acyl group of the parent compound;

omission of the 5-methyl substituent from the acyl group of the sidechain; and

omission of the acyl group of the side chain.

In particular, the mimic is preferably a kahalalide F derivative whichis not a mix of isomers known as kahalalide F. Such a derivative canhave a structure with a cyclic part and a side chain derived from theformula (I):

the derivative differing from the formula (I) in one or more of thefollowing respects:

1 or 2 amino acids which are not the same as an amino acid in thestructure of formula (I);

1 to 10 additional methylene groups in the acyl group of the side chainof the structure of formula (I);

1 to 5 methylene groups omitted from the acyl group of the side chain ofthe structure of formula (I);

1 to 3 substituents added to the side chain acyl group of the structureof formula (I);

omission of the 5-methyl substituent from the acyl group of the sidechain; and

omission of the acyl group of the side chain.

For example, the compound can differ from the formula (I) in one or moreof the following respects:

1 amino acid which is not the same as an amino acid in the structure offormula (I);

1 additional methylene group in the side chain acyl group of thestructure of formula (I);

1 methylene group omitted from the side chain acyl group of thestructure of formula (I);

1 substituent added to the side chain acyl group of the structure offormula (I);

omission of the 5-methyl substituent from the acyl group of the sidechain.

The 1 or 2 amino acids which are not the same as an amino acid in thestructure of formula (I) can be omitted amino acids. There can beomission from the cyclic part of the structure.

In one aspect, each amino acid is as in formula (I). The side chain canbe a congener of 5-MeHex-D-Val-L-Thr-L-Val-D-Val-D-Pro-L-Orn-D-allo-Ile.For instance, the 5-MeHex can be replaced by a terminal alkyl, alkenyl,alkynyl, aryl, aralkyl, heteroaryl, heteroalkyl, or alicyclic group,especially a terminal alkyl group. Such a group can have 4 to 10 carbonatoms. The terminal alkyl group suitably has 1 or more methyl or ethylgroups branching distal to the point of attachment to the rest of themolecule, and preferably a single branched methyl group.

The terminal alkyl group can be substituted with one or more halogen,hydroxy, alkoxy, amino, carboxyl, carboxamido, cyano, nitro,alkylsulfonyl, alkoxy, alkoxyalkyl, arylalkylaryl, heterocyclic,alicyclic, aryl or aralkyl groups.

Chirality can be present in the replacement terminal group, and theinvention embraces the individual isomers as well as mixes thereofincluding racemic mixes.

More details of preferred definitions and typical compounds are given inthe texts incorporated herein by reference, WO 01 58934 and WO2005023846. We currently prefer that the terminal acyl group in thesidechain is 5-methylhexanoyl, 4-methylhexanoyl or more especially4(S)-methylhexanoyl. Especially preferred is a mimic which is of theformula given above for kahalalide F but has a 4(S)-methylhexanoylgroup.

The process of this invention involves coupling a cyclic part with aside chain fragment. The cyclic part can itself contain at least one ofthe side chain amino acids. Alternatively the side chain fragment cancorrespond to the complete side chain of the desired compound.

The cyclic part is preferably that of kahalalide F or of a mimic definedabove, and can contain one or more amino acids that are present in theside chain of the desired compound. It is preferred that the directproduct of the coupling is an optionally protected form of the desiredend product, kahalalide F or a mimic. Thus the preferred reactionsconsist of the coupling step and then deprotection to give the desiredcompound.

DETAILED DESCRIPTION OF THE INVENTION

We have identified new synthetic routes to kahalalide compounds. Theseare based in convergent approaches using orthogonal protecting schemes,where a better control of the intermediates is taken.

Convergent strategies are defined as those in which peptide fragmentsare coupled together to give the desired target molecule. Thecondensation of peptide fragments should lead to fewer problems in theisolation and purification of intermediates. The difference between thedesired condensation product and the segments themselves, in terms ofmolecular size and chemical nature, should be sufficiently pronounced soas to permit their separation relatively easily (Lloyd-Williams, P.;Albericio, F.; Giralt, R. “Chemical approaches to the synthesis ofpeptides and proteins”. CRC Press. Boca Raton (FL), 1997).

An orthogonal protecting scheme has been defined as one based oncompletely different classes of protecting groups such that each classof groups can be removed in any order and in the presence of all otherclasses of protecting group (Barany, G.; Albericio, F. J. Am. Chem. Soc,107, 4936 (1985)).

Preparation of protected peptides can be carried out in solution and/orin solid-phase, as well as from natural kahalalide F isolated fromeither the mollusc or the alga or obtained by fermentation. Assemblingof the protected peptides are preferably carried out in solution. Allintermediates can be characterized and, if needed, purified.

Examples of strategies covered by this invention are shown in thefollowing Scheme I:

wherein AAA is an aliphatic/fatty acid or an aliphatic/fatty acylaminoacid or a peptide and BBB, CCC, DDD and EEE are amino acids or peptides.Amino acids are independently selected from natural or non-natural aminoacids of L or D configuration, if applies; CCC should contain atrifunctional amino acid capable of forming a covalent bond, preferablyester, thioester, or amide, with the carboxyl function of the C-terminalamino acid of peptide EEE. CCC, DDD, and EEE form part of a cycle, andthe process involves extending the exocyclic chain.

In a preferred aspect, the invention involves a synthetic strategyconsisting of adding amino acids BBB of the exocylic chain to the cyclicpart of the compound where CCC, DDD, and EEE form the cycle. The aminoacids can be added one by one, or in fragments that contain two or moreamino acids including the terminal aliphatic/fatty acid AAA.

In a preferred synthesis, two fragments are separately constructed forexample using solid phase synthesis, one containing at least the cyclicpart optionally with one or more of the sidechain amino acids, and onecontaining at least the terminal acid attached to one or more sidechainamino acids. The fragments can be cleaved from the solid phase andjoined.

In particular, we can consider the synthesis of compounds of formula(1):

wherein RCO is a terminal acyl and R is preferably a branched alkylgroup, especially an alkyl group of 6 to 8 carbon atoms and with asingle methyl branch. Examples of RCO are 5-methylhexanoyl or4(S)-methylhexanoyl.

The most preferred strategy involves making the join between D-Pro-9 andL-Orn-8. There are other candidate strategies that are possible such asL-Orn-8 and D-allo-Ile-7, and also D-Val-10 and D-Pro-9, D-allo-Ile-7and D-allo-Thr-6 or L-Val- 1 and D-Val-10.

Following the coupling of all the molecule it is also possible to carryout dehydration and deprotection reactions in order to obtain thedesired final compound.

Another possibility for generating the fragment including the cyclicpart is to start with natural KF, and treat it with trypsin (it will cutby the Orn). It is then possible to perform the coupling with thenatural fragment and a non natural fragment (the one with the acylgroup).

The preferred embodiment of the synthetic process of the presentinvention is best represented in the Scheme II, which is directed to theformation of the target compound with a 4(S)-methylhexyl terminal acid.

The key steps of the optimized process for a more economical and safesynthesis of kahalalide analogues are: (i) preparation of the twoprotected peptides onto a chlorotritylchloro-polystyrene resin orrelated resin; (ii) the C-terminal residue at the N-terminal protectedpeptide is Pro, which is the least prone to be racemized during thecoupling reagent; (iii) for the stepwise synthesis of both protectedpeptides, use of DIPCDIC-HOBt as coupling method instead of HATU-DIPEA,for the sequential incorporation of the protected amino acids andaliphatic carboxylic acids; (iv) use of sodium diethyl-dithiocarbamateafter removing Alloc to avoid presence of Pd (0) in the final product;(v) cyclization step with DIPCDI/HOBt/DIPEA in CH₂Cl₂; these conditionsavoids two side reaction: epimerisation of the Val residue, which isinvolved in the activation, and trifluoroacetylation of the Phe or itsreplacement; (vi) removal of the Fmoc group in such conditions thatleaves inalterated the cyclic; (vii) if needed, purification of bothprotected peptides; (viii) coupling of both protected peptides withPyAOP/DIEA.

As shown above in Scheme II, the preferred process for the syntheticformation of analogues of Kahalalide F is based in a convergentsolid-phase (protected fragment syntheses) and solution (cyclization,fragment condensation, and final deprotection) method using anorthogonal protecting scheme based on a Fmoc/tBu strategy, see forexample Lloyd-Williams, P., et al. Chemical Approaches to the Synthesisof Peptides and Proteins. CRC Press, Boca Raton (FL), 1997.

The process of Scheme II comprises the sequential steps of:

Protected Fragment I

(a) incorporating an Fmoc-DVal-OH onto a chlorotrityl chloro resin,forming an ester bond;

(b) elongating the peptidic chain with three amino acids [Dalle, DaThr(free OH), DaIle) using a Fmoc/tBu strategy;

(c) incorporating [Val(l)] using an Alloc as protecting group;

(d) incorporating Fmoc-Orn(Boc)-OH;

(e) incorporating the dipeptide Alloc-Phe-ZDhb-OH, which has beencombined and dehydrated in solution;

(f) removing the Alloc, or of its replacement, while the peptide isstill anchored to the solid support;

(g) cleaving the side-chain protected peptide from the solid support;

(h) cyclizing the peptide in solution;

(i) removing the Fmoc group of the Orn.

Alternatively, protected fragment can be prepared starting by

(a′) incorporating Alloc-Phe-ZDhb-OH, which has been combined anddehydrated in solution, onto a chlorotrityl chloro resin, forming anester bond;

(b′) elongating the peptidic chain with five amino acids [Fmoc-DVal-OH,Frmoc-DaIle-OH, Fmoc-DaThr-OH (free OH), Fmoc-DaIle-OH, andAlloc-Orn(Boc)-OH];

(c′) incorporating Val(1) using a Fmoc as protecting group;

(d′) removing the Fmoc while the peptide is still anchored to the solidsupport;

(e′) cleaving the side-chain protected peptide from the solid support;

(g′) cyclizing the peptide in solution;

(h′) removing the Alloc group of the Orn.

This alternate strategy shows the following advantages:

(i) as the incorporation onto a chlorotrityl chloro resin requires adefect of protected building block, the amount of the preciousAlloc-Phe-ZDhb-OH used is less if compared with the first strategy,

(ii) possibility of preparing Fmoc-Phe-Z-Dhb-OH (avoiding a Pdtreatment),

(iii) minimization or removal of racemization during the cyclization,and

(iv) stability of cycle to Pd(0) used to remove the Alloc (step h′).

Protected Fragment II

(a) incorporating an Fmoc-DPro-OH onto a chlorotrityl chloro resin,forming an ester bond;

(b) elongating the peptidic chain using a Fmoc/ tBu strategy;

(c) cleaving the side-chain protected peptide from the solid support;

Final Steps

(i) fragment condensation

(ii) final deprotection

Therefore the process can be conducted as follows:

Fmoc-DVal-OH or Alloc-Phe-ZDhb-OH, which was prepared in solution fromAlloc-Phe-OH and H-Thr-OtBu with EDC,HCl, and posterior dehydration andtreatment with TFA, are incorporated preferably to achlorotrityl-polystyrene resin, see Barlos, K.; Gatos, D.; Schäfer, W.Angew. Chem. Int. Ed. Engl. 1991, 30, 590-593.

Removal of the Fmoc group can be carried out with piperidine-DMF (2:8,v/v) (1×2 min, 2×10 min). Couplings of Fmoc-aa-OH (4-5 equiv) (aa meansamino acid) can be carried out with DIPCDI-HOBt (equimolar amounts ofeach one respect to the carboxylic component) or PyBOP-DIPEA (equimolaramount of PyBOP and double amount of DIPEA) in DMF or DMF-Toluene (1:1)for 90 min. After the coupling ninhydrin or chloranil tests are carriedout and if it is positive the coupling is repeated in the sameconditions, otherwise the process is continued. Washings betweendeprotection, coupling, and, again, deprotection steps can be carriedout with DMF (5×0.5 min) and CH₂Cl₂ (5×0.5 min) using for example eachtime 10 mL solvent/g resin.

Incorporation of Alloc-Val-OH (5 equiv) can be carried out withequimolar amount of DIPCDI and 10% of DMAP. This coupling is repeated atleast twice.

Removal of Alloc group can be carried out with Pd(PPh₃)₄ (0.1 equiv) inthe presence of PhSiH₃ (10 equiv), see Gómez-Martinez P., Thieriet N.,Albericio F., Guibé F. J. Chem. Soc. Perkin I 1999, 2871-2874, andwashing the resin with sodium diethyldithiocarbamate in DMF 0.02 M (3×15min).

In the first strategy, the dipeptide Alloc-Phe-ZDhbOH (4 equiv), can becoupled to the Val (1) residue anchored to the resin with DIPCDI-HOAt (4equiv of each) for 5 h to overnight.

Cleavage of the protected peptide from the resin can be accomplished byTFA-CH₂Cl₂ (1:99) (5×30 sec).

Cyclization step can be carried out with DIPCDI/HOBt/DIPEA in CH₂Cl₂.These conditions avoid two side reactions: epimerisation of the Valresidue, which is involved in the activation, and trifluoroacetylationof the Phe or its replacement.

In the first strategy, removal of the Fmoc group of the Orn residue with20 equiv of dimethylamine minimize the opening of the cyclic.

Fragment condensation is carried out with PyAOP/DIPEA (equimolar amountof PYAOP with respect to carboxylic component and 3 equivalents ofDIPEA). After 1 hour, is used another equimolar amount of PyAOP is used(until the condensation is completed).

Final deprotection can be carried out with TFA-H₂O (95:5) for 1 h.

It will be appreciated that the particular choice of protecting groupsis not critical, and other choices are widely available. For example,Bzl type groups can replace tBu/Boc; Boc instead of Fmoc; pNZ instead ofFmoc of Orn; Fmoc instead of Alloc; Alloc instead of Fmoc; Wang resininstead of chlorotrityl. Those protecting groups and resins aredescribed in Greene and Wuts, Protective Groups in Organic Synthesis,John Wiley & Sons, Inc, New York, 1999 and Goodman, M. ed. Houben-Weyl,Methods of Organic Chemistry, Vol. E 22A, Synthesis of Peptides andPeptidomimetics, Thieme, Stuggart-New York, 2001.

Further detail on the synthesis is given in the examples.

The process of this invention can be carried out from starting materialsin an enantio-, stereocontrolled and fast manner, taking advantages ofthe solid-phase synthetic methodology, where the molecule inconstruction is bounded to an insoluble support during all syntheticoperations.

EXAMPLES

General Procedures. Cl-TrtCl-resin, Protected Fmoc-amino acidderivatives, HOBt, HOAt were from ABI (Framingham, Mass.), Bachem(Bubendorf, Switzerland), NovaBiochem (Läufelfingen, Switzerland), andIRIS Biotech (Marktredwitz, Germany). 4(S)-MeHex derivatives fromNarchem.

Alloc-amino acids were prepared essentially as described by Dangles etal. (see J. Org. Chem. 1987, 52, 4984-4993) and Cruz et al. (see Org.Proc. Res. Develop. 2004, 8, 920-924). Alloc-ZDhb-Phe-OH was prepared asdescribed in WO 01 58934, and DIPEA, DIPCDI, EDC-HCl, Piperidine, TFAwere from Aldrich (Milwaukee, Wis.). DMF and CH₂Cl₂ were from SDS(Peypin, France). ACN (HPLC grade) was from Scharlau (Barcelona, Spain).All commercial reagents and solvents were used as received withexception of CH₂Cl₂, which was passed through a alumina column to removeacidic contaminants.

Solid-phase syntheses were carried out in polypropylene syringes (10-50mL) fitted with a polyethylene porous disc. Solvents and solublereagents were removed by suction. Removal of the Fmoc group was carriedout with piperidine-DMF (2:8, v/v) (1×2 min, 2×10 min). Washings betweendeprotection, coupling, and, again, deprotection steps were carried outwith DMF (5×0.5 min) and CH₂Cl₂ (5×0.5 min) using each time 10 mLsolvent/g resin. Peptide synthesis transformations and washes wereperformed at 25° C. Synthesis carried out on solid-phase were controlledby HPLC of the intermediates obtained after cleaving with TFA-H20 (1:99)for 1 min an aliquot (aprox. 2 mg) of the peptidyl-resin. HPLC reversedphase columns Symmetry™ C₁₈ 4,6×150 mm, 5 μm (column A) and Symmetry300™C₁₈ 4,6×50 mm, 5 μm (column B) were from Waters (Ireland). AnalyticalHPLC was carried out on a Waters instrument comprising two solventdelivery pumps (Waters 1525), automatic injector (Waters 717autosampler), dual wavelength detector (Waters 2487), and systemcontroller (Breeze V3.20) and on a Agilent 1100 instrument comprisingtwo solvent delivery pumps (G1311A), automatic injector (G1329A), DAD(G1315B). UV detection was at 215 or 220 nm, and linear gradients ofCH₃CN (+0.036% TFA) into H₂O (+0.045% TFA), from 30% to 100% in 15 min.MALDI-TOF and ES-MS analysis of peptide samples were performed in aPerSeptive Biosystems Voyager DE RP, using ACH matrix, and in a WatersMicromass ZQ spectrometer and in an Agilent Ion Trap 1100 SeriesLC/MSDTrap. Peptide-resin samples were hydrolyzed in 12 N aqueousHCl-propionic acid (1:1), at 155° C. for 1-3 h and peptide-free sampleswere hydrolyzed in 6 N aqueous HCl at 155° C. for 1 h. Subsequent aminoacid analysis was performed on a Beckman System 6300 autoanalyzer.

Nomenclature used for cyclic peptides and precursors as described bySpengler et al. (see Spengler J., Jimenez J. C., Burger K., Giralt E.,Albericio, F. “Abbreviated nomenclature for cyclic and branched homo-and hetero-detic peptides”. J. Peptide Res. 2005, on line publication:13-Apr-2005). The “&” symbol is used in the nomenclature for cyclicpeptides and precursors. The appearance of “&,” in a given position ofthe one-line formula represents the point at which one end of a chemicalbond is located and the second “&” indicates the point to which thisbond is attached. Thus, “&” represents the start or the end of achemical bond, which is ‘cut’ with the aim of visualizing a complexformula more easily. In this way, two “&” symbols represent one chemicalbond.

Example 1[H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-OH][H-Phe-(Z)Dhb-Val&]from Fmoc-Orn(Boc)-OH Step 1

H-D-Val-O-TrtCl-resin.

Cl-TrtCl-resin (1 g, 1.64 mmol/g) was placed in a 20 mL polypropylenesyringe fitted with a polyethylene filter disk. The resin was thenwashed with CH₂Cl₂ (5×0.5 min), and a solution of Fmoc-D-Val-OH (238 mg,0.7 mmol, 0.43 equiv) and DIPEA (0.41 mL) in CH₂Cl₂ (2.5 mL) was added,and the mixture was stirred for 15 min. Then extra DIPEA (0.81 mL, total7 mmol) was added and the mixture was stirred during 45 min more. Thereaction was terminated by addition of MeOH (800 μL), after a stirringof 10 min. The Fmoc-D-Val-O-TrtCl-resin was subjected to the followingwashings/treatments with CH₂Cl₂ (3×0.5 min), DMF (3×0.5 min), piperidineas indicated in General Procedures, and DMF (5×0.5 min). The loadingcalculated by Fmoc determination was 0.50 mmol/g.

Step 2

[Fmoc-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-TrtCl-resin][Alloc-Val&].

Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv), Fmoc-D-allo-Thr-OH (freehydroxy group) (683 mg, 2 mmol, 4 equiv), and Fmoc-D-allo-Ile-OH (707mg, 2 mmol, 4 equiv) were added sequentially to the above obtainedH-D-Val-O-TrtCl-resin using DIPCDI (310 μL, 2 mmol, 4 equiv) and HOBt(307 mg, 2 mmol, 4 equiv) in DMF (2.5 mL). In all cases, after 90 min ofcoupling, the ninhydrin test was negative. Removal of Fmoc group andwashings were carried out as described in General Procedures.Alloc-Val-OH (502 mg, 2.5 mmol, 5 equiv) was coupled with DIPCDI (387mg, 2.5 mmol, 5 equiv) in the presence of DMAP (30.6 mg, 0.25 mmol, 0.5equiv) and DIPEA (88 μL, 0.5 mmol, 1 equiv) for 45 min. This couplingwas repeated in the same conditions twice. An aliqout of thepeptidyl-resin was treated with TFA and the HPLC (t_(R) 14.2 min, columnA) of the crude obtained after evaporation showed a purity of >98%.

ESMS, calcd for C₄₅H₆₃N₅O1 ₁, 849.45. Found: m/z 850.1 [M+H]⁺, 871.9[M+Na]⁺.

Step 3

[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-TrtCl-resin][Alloc-Val&,].

The Fmoc group of the above peptidyl-resin (Step 2) was removed andFmoc-Orn(Boc)-OH (912 mg, 2 mmol, 4 equiv) was added using DIPCDI (310μL, for 2.0 mmol and 4 equiv; and 388 μL, for 2.5 mmol and 5 equiv) andHOBt (307 mg, for 2.0 mmol and 4 equiv; and 395 mg, for 2.5 mmol and 5equiv) for 90 min. Ninhydrin test after the incorporation was negative.An aliqout of the peptidyl-resin was treated with TFA and the HPLC(t_(R) 12.8 min, column A) of the crude obtained after evaporationshowed a purity of 90%.

ESMS, calcd for C₅₆H₈₁N₇O₁₄, 1,063.58. Found: m/z 1,086.77 [M+Na⁺]⁺.

Step 4

[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-TrtCl-resin][Alloc-Phe-ZDhb-Val&,].

Alloc group of the above peptidyl-resin (Step 3) was removed withPd(PPh₃)₄ (58 mg, 0.05 mmol, 0.1 equiv) in the presence of PhSiH₃ (617μL, 5 mmol, 10 equiv), followed by washings with diethyldithiocarbamate0.02 M (3×15 min). Alloc-Phe-ZDhb-OH (666 mg, 2 mmol, 4 equiv) and HOAt(273 mg, 2 mmol, 4 equiv) were dissolved in DMF (1.25 mL) and added topeptidyl-resin. Then DIPCDI (310 μL, 2 mmol, 4 equiv) was added and themixture stirred for 5 h, where the ninhydrin test was negative. Afterwashings with DMF and CH₂Cl₂, an aliquot of the peptidyl-resin wastreated with TFA-H₂O (1:99) for 1 min and the product was characterizedby MALDI-TOF-MS.

MALDI-TOF-MS, calcd for C₆₈H₉₅N₉O₁₆, 1,293.69 Found: m/z 1,294.35[M+H]⁺, 1,316.39 [M+Na]⁺, 1,333.34 [M+K]⁺.

Step 5

[Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-OH][H-Phe-ZDhb-Val&].

Alloc group of the above peptidyl-resin (Step 4) was removed withPd(PPh₃)₄ (58 mg, 0.05 mmol, 0.1 equiv) in the presence of PhSiH₃ (617μL, 5 mmol, 10 equiv), the resin was washed with sodiumdiethyldithiocarbamate in DMF 0.02 M (3×15 min) and the protectedpeptide was cleaved from the resin by TFA-CH₂Cl₂ (1:99) (5×30 sec).Filtrate was collected on H₂O (4 mL) and the H₂O was partially removedunder reduced pressure. ACN was then added to dissolve solid thatappeared during the H₂O removal, and the solution was lyophilized, togive 700 mg (578 μmol, 99% yield of the title compound with a purityof >91% as checked by HPLC (Column A, t_(R) 8.59 min).

MALDI-TOF-MS, calcd for C₆₄H₉₁N₉O₁₄, 1,209.67. Found: m/z 1,210.45[M+H]⁺, 1,232.51 [M+Na]⁺, 1,248.45 [M+K]⁺.

Step 6

Fmoc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&.

The protected peptide (Step 5) (700 mg, 578 μmol) was dissolved inCH₂Cl₂ (580 mL, 1 mM), and HOBt (137 mg, 2.3 mmol) dissolved in theminimum volume of DMF to dissolve HOBt, DIPEA (302 μL, 1.73 mmol, 3equiv), and DIPCDI (356 μL, 2.3 mmol, 4 equiv) were added. The mixturewas allowed to stir for 1 h, and then the course of the cyclization stepwas checked by HPLC (column A, t_(R) 12.4 min). The solvent was removedby evaporation under reduced pressure.

MALDI-TOF-MS, calcd for C₆₄H₈₉N₉O₁₃, 1,191.66. Found: m/z 1,092.17[M+H]⁺, 1,214.14 [M+Na]⁺, 1,230.10 [M+K]⁺.

Step 7

H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

The protected peptide (Step 6) (50 mg, 42 μmol) was dissolved in DMF (5mL), then diethylamine (130 μL, 30 equiv) was added and the mixture wasleft to stir by 1:30 min. The solvent was removed by evaporation underreduced pressure. The crude product was purified by HPLC (Symmetry C₈ 5[μm, 30×100 mm), linear gradient of ACN (30% to 75% in 15 min) ACN(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220 nm. Theproduct was characterized by HPLC (t_(R) 8.7 min, Condition A) and forMALDI-TOF-MS, calcd C₄₉H₇₉N₉O₁₁, 969.59. Found: m/z 970.87 [M+H]⁺,870.78 [M-Boc]⁺.

Example 2[H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-OH][H-Phe-(Z)Dhb-Val&]from NZ-Orn(Boc)-OH

Experimental procedures as described in Examples 1, except that in thestep 3, Fmoc-Orn-OH is replaced by pNZ-Orn-OH.

The protected peptide (14.7 mg, 12.8 μmol) was dissolved in 1.6 mM HClin DMF (10 mL), then SnCl₂ (3.8 g, 20 mmol) was added and the mixturewas left to stir until HPLC (Column A) showed the completion of thereaction (1 h). The solvent was removed by evaporation under reducedpressure. The crude product was purified by HPLC (Symmetry C₈ 5 μm,30×100 mm), gradient of ACN (30% to 75% in 15 min) ACN (+0.05% TFA) inwater (+0.05% TFA), 20 mL/h, detection at 220 nm, to give the titleproduct (4.8 mg, 4.9 pmol, 40% yield. The product was characterized byHPLC (t_(R) 8.2 min, Column A) and for MALDI-TOF-MS.

MALDI-TOF-MS, calcd C₄₉H₇₉N₉O₁₁, 969.59. Found: m/z 992.35 [M+Na]⁺,870.34 [M-Boc]⁺, 892.34 [M+Na-Boc]⁺.

Example 3[H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-OH][H-Phe-(Z)Dhb-Val&]from Alloc-Orn(Boc)-OH Step 1

H-Phe-(Z)Dhb-O-TrtCl-resin.

Cl-TrtCl-resin (1 g, 1.64 mmol/g) was placed in a 20 mL polypropylenesyringe fitted with a polyethylene filter disk. The resin was thenwashed with CH₂Cl₂ (5×0.5 min), and a solution of Alloc-Phe-(Z)Dhb-OH(232 mg, 0.7 mmol, 0.42 equiv) and DIPEA (0.41 mL) in CH₂Cl₂ (2.5 mL)was added, and the mixture was stirred for 15 min. Then extra DIPEA(0.81 mL, total 7 mmol) was added and the mixture was stirred during 45min more. The reaction was terminated by addition of MeOH (800 μL),after a stirring of 10 min. The Alloc-Phe-(z)Dhb-O-TrtCl-resin wassubjected to the following washings with CH₂Cl₂ (3×0.5 min), DMF (3×0.5min), and the Alloc group was removed with Pd(PPh₃)₄ (58 mg, 0.05 mmol,0.1 equiv) in the presence of PhSiH₃ (617 μL, 5 mmol, 10 equiv) inCH₂Cl₂. The resin was washed as described in General Procedures. Theloading calculated by Fmoc determination was 0.68 mmol/g.

Step 2

[Alloc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-(Z)Dhb-OH][H-Val&]

Fmoc-D-Val-OH (678 mg, 2 mmol, 4 equiv), Fmoc-D-allo-Ile-OH (707 mg, 2mmol, 4 equiv), Fmoc-D-allo-Thr-OH (free hydroxy group) (683 mg, 2 mmol,4 equiv), Fmoc-D-allo-Ile-OH (707 mg, 2 mmol, 4 equiv), andAlloc-Orn(Boc)-OH (630 mg, 2 mmol, 4 equiv) were added sequentially tothe above obtained H-Phe-(Z)Dhb-O-TrtCl-resin using DIPCDI (310 μL, 2mmol, 4 equiv) and HOBt (307 mg, 2 mmol, 4 equiv) in DMF (2.5 mL). Inall cases, after 90 min of coupling, the ninhydrin test was negative.Removal of Fmoc group and washings were carried out as described inGeneral Procedures. Fmoc-Val-OH (848.2 mg, 2.5 mmol, 5 equiv) wascoupled with DIPCDI (387 mg, 2.5 mmol, 5 equiv) in the presence of DMAP(30.6 mg, 0.25 mmol, 0.5 equiv) and DIPEA (88 μL, 0.5 mmol, 1 equiv) for45 min. This coupling was repeated in the same conditions twice. Afterremoval of the Fmoc group as described in General Procedures, theprotected peptide was cleaved from the resin by TFA-CH₂Cl₂ (1:99) (5×30sec). Filtrate was collected on H₂O (4 mL) and the H₂O was partiallyremoved under reduced pressure. ACN was then added to dissolve the solidthat appeared during the H₂O removal, and the solution was lyophilized,to give 650 mg (606 μmol, 90% yield of the title compound with a purityof >75% as checked by HPLC (Column A, t_(R) 9.93 min).

ESMS, calcd for C₅₃H₈₅N₉O₁₄, 1072, 29. Found: m/z 1074,4 [M+H]⁺.

Step 3

Alloc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&.

The protected peptide (Step 2) (250 mg, 0.233 mmol) was dissolved inCH₂Cl₂ (240 mL, 1 mM), and HOAt (126 mg, 9.325 mmol, 4 equiv) dissolvedin the minimum volume of DMF to dissolve HOAt, and DIPCDI (143 μL, 9,325mmol, 4 equiv) were added. The mixture was allowed to stir for 24 h,then the course of the cyclization step was checked by HPLC (column A,t_(R) 12.82 min). The solvent was removed by evaporation under reducedpressure.

MALDI-TOF-MS, calcd for C₅₃H₈ ₃N₉O₁₃, 1,054.28. Found: m/z 1056.4[M+H]⁺.

Step 4

H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&.

The protected peptide (Step 3) (244 mg, 231 μmol) was dissolved in 10 mLof CH₂Cl₂, then Pd(PPh₃)₄ (8 mg, 6.94 μmol, 0.03 equiv) in the presenceof PhSiH₃ (94 μL, 763.6 μmol, 3.3 equiv) was added and the mixture wasleft to stir by 1:30 min. The solvent was removed by evaporation underreduced pressure. The crude product was purified by HPLC (Symmetry C₈ 5Mm, 30×100 mm), linear gradient of ACN (20% to 80% in 15 min) ACN(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220 nm. Theproduct was characterized by HPLC (t_(R) 9.19 min, Condition A) and forMALDI-TOF-MS, calcd C₄₉H₇₉N₉O₁₁, 970.21. Found: m/z 972.1 [M+H]⁺.

Example 4 (4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-OH Step 1

H-D-Pro-O-TrtCl-resin.

Cl-TrtCl-resin (1 g, 1.64 mmol/g) was placed in a 20 mL polypropylenesyringe fitted with a polyethylene filter disk. The resin was thenwashed with CH₂Cl₂ (5×0.5 min), and a solution of Fmoc-D-Pro-OH (237 mg,0.7 mmol, 0.43 equiv) and DIPEA (0.41 mL) in CH₂Cl₂ (2.5 mL) was added,and the mixture was stirred for 15 min. Extra DIPEA (0.81 mL, total 7mmol) was added and the mixture was stirred for 45 min. The reaction wasterminated by addition of MeOH (800 μL), after a stirring of 10 min. TheFmoc-D-Pro-O-TrtCl-resin was subjected to the followingwashings/treatments with CH₂Cl₂ (3×0.5 min), DMF (3×0.5 min), piperidineas indicated in General Procedures, and DMF (5×0.5 min). The loadingcalculated by Fmoc determination was 0.27 mmol/g.

Step 2

(4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-OH

Fmoc-D-Val-OH (458 mg, 1.32 mmol, 5 equiv), Fmoc-Val-OH (360 mg, 1.06mmol, 4 equiv), Fmoc-Thr(tBu)-OH (527 mg, 1.32 mmol, 5 equiv),Fmoc-D-Val-OH (360 mg, 1.06 mmol, 4 equiv), and (4S-MeHex-OH (138 mg,1.06 mmol, 4 equiv) were sequentially added to the above peptidyl-resin(Step 1) using DIPCDI (165 μL, for 1.06 mmol and 4 equiv; and 205 μL,for 1.32 mmol and 5 equiv) and HOBt (162 mg, for 1.06 mmol and 4 equiv;and 203 mg, for 1.32 mmol and 5 equiv) for 90 min. In all cases, after90 min of coupling, the ninhydrin test was negative. Removal of Fmocgroup and washings were carried out as described in General Procedures.

The partial protected peptide was cleaved from the resin by TFA-CH₂Cl₂(1:99) (5×30 sec). Filtrate was collected on H₂O (4 mL) and the H₂O waspartially removed in a rotavapor. ACN was then added to dissolve thesolid that appeared during the H₂O removal, and the solution waslyophilized, to give 154.4 mg (226 μmol, 85.5% yield) of the titlecompound with a purity of >94% as checked by HPLC (Column A, t_(R) 12.13min). The crude obtained after evaporation showed a purity of >94%. Theproduct was characterized by Electrospray.

Calcd for C₃₅H₆₃N₅O₈, 681.9. Found: m/z 682.15.

Example 5(4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

Peptides from Examples 1 (8.25 mg, 8.5 μmol) and 4 (7 mg, 10.2 μmol, 1.2equiv) were dissolved in DMF (10 mL) and PyAOP (5.32 mg, 10.2 μmol, 1.2equiv) and DIPEA (5.3 μl, 30.6 μmol, 3.6 equiv) were added at roomtemperature. The mixture was stirred for 1 h, when extra PyAOP (5.32 mg,10.2 μmol, 1.2 equiv) were added. The mixture was allowed to react for 2h at room temperature, until HPLC (Column A) showed the completion ofthe reaction. The crude obtained after evaporation showed by HPLC apurity of >75%. The crude product was purified by HPLC (Symmetry C₈ 5μm, 30×100 mm), linear gradient of ACN (+0.05% TFA) in water (+0.05%TFA) (30% to 100% in 15 min), 20 mL/h, detection at 220, to give thetitle product (6.9 mg, 4.2 μmol, 49% yield).

MALDI-TOF-MS, calcd for C₈ ₄H₁₄₀N₁₄O₁₈, 1.633.05. Found: m/z 1,534.33[M-Boc]⁺,1,556.26 [M-Boc+Na]⁺ 1,656.33 [M+Na]⁺.

Example 6(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

The protected cyclic peptide (Example 5) was dissolved in TFA-H₂O (19:1,700 μL) and the mixture was allowed to stir for 1 h. The solvent wasremoved by evaporation under reduced pressure, and dioxane was added(245 μL). The solvent was removed by evaporation under reduced pressure(the process was repeated three times), and then H₂O (1 mL) was addedand lyophilized. The crude product was purified by HPLC (Symmetry C₈ 5μm, 30×100 mm), isocratic 44% ACN (+0.05% TFA) in water (+0.05% TFA), 20mL/h, detection at 220 nm, to give the title product (5 mg, 3.4 μmol,80% yield, 93.3%). The HPLC did not show the presence of the epimericpeptide(4S)-MeHex-D-Val-Thr-Val-D-Val-Pro-Orn-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&(Example 7), which would indicate racemization during the coupling stepbetween both protected peptides.

MALDI-TOF-MS, calcd for C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,478.17[M+H]⁺ 1,500.14 [M+Na]⁺, 1,516.12 [M+K]⁺.

Example 7(4S)-MeHex-D-Val-Thr-Val-D-Val-Pro-Orn-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

Experimental procedures as described in Examples 1,4,5 and 6, exceptthat in the step 1 of Example 4 Fmoc-D-Pro-OH is replaced byFmoc-Pro-OH. The product was characterized by HPLC (t_(R) 11.23 min,Column A) and for MALDI-TOF-MS.

MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,500.23[M+Na]⁺, 1,515.97 [M+K]⁺.

Example 8 H-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-(Z)Dhb-Val&

Starting with[Fmoc-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-O-TrtCl-resin][Alloc-Val&](Step 2, Example 1), the Alloc group was removed with Pd(PPh₃)₄ (58 mg,0.05 mmol, 0.1 equiv) in the presence of PhSiH₃ (617 μL, 5 mmol, 10equiv) and the resin washed with sodium diethyldithiocarbamate in DMF0.02 M (3×15 min). Alloc-Phe-Z-Dhb-OH (666 mg, 2 mmol, 4 equiv) and HOAt(273 mg, 2 mmol, 4 equiv) were dissolved in DMF (1.25 mL) and added topeptidyl-resin. Then DIPCDI (310 μL, 2 mmol, 4 equiv) was added and themixture stirred for 5 h, when the HPLC showed the completion of reaction(t_(R) 7.09 min, Column A).

The Fmoc group was removed and after extensive DMF washings, Boc₂O (546mg, 5 equiv) and DIPEA (0.87 mL, 10 equiv) in DMF were added and left toreact for 2 h, when the ninhydrin test was negative. After DMF washings,the Alloc group was removed as above and the protected peptide wascleaved from the resin with TFA-CH₂Cl₂ (1:99) (5×30 sec). Filtrate wascollected on H₂O (4 mL) and the H₂O was partially removed under reducedpressure. ACN was then added to dissolve solid that appeared during theH₂O removal, and the solution was lyophilized.

Cyclization was carried out as in Step 6 of Example 1; and then the Bocwas removed with TFA-H₂O (19:1) (1 h). The solvent was removed underreduced pressure and dioxane was added (245 μL). The solvent was removedby evaporation under reduced pressure (the process was repeated threetimes), and then H₂O (1 mL) was added and lyophilized. The crude productwas purified by HPLC (Symmetry C₈ 5 μm, 30×100 mm), isocratic 44% ACN(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220 nm, togive the title product (207 mg, 273 μmol, 55% yield, 93.3%).

The product was characterized by HPLC (t_(R) 7.27 min, Column A) and forMALDI-TOF-MS.

MALDI-TOF-MS, calcd C₃₉H₆₁N₇O₈, 755.46. Found: m/z 756.56 [M+H]⁺, 778.55[M+Na]⁺, 794.53 [M+K]⁺.

Example 9 (4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-Orn(Boc)-OH

Experimental procedures as described in Example 4, except that thepeptide synthesis was initiated by incorporation of Fmoc-Orn(Boc)-OH tothe Cl-TrtCl-resin. The product was characterized by HPLC (t_(R) 13.27min, Column A) and for Electrospray.

Calcd C₃₆H₆₅N₇O₉, 739.48. Found: m/z 740.65.

Example 10(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D-allo-lie-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

Experimental procedures as described in Examples 5 and 6, except that inExample 5 peptide from Example 1 is replaced by peptide from Example 8and peptide from Example 4 is replaced by peptide from Example 9. TheHPLC of the final product showed the presence of the epimeric peptide(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-D-Orn-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&(4.1%) (Example 11), which indicates racemization during the couplingstep between both protected peptides. The product was characterized byHPLC (t_(R) 10.5 min, Column A).

MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,477.99 [M+H]⁺1,499.97 [M+Na]⁺, 1,515.93 [M+K]⁺.

Example 11(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-D-Orn-D-allo-Ile-D-atio-Thr(&)-D-aZlo-Ile-D-Val-Phe-ZDhb-Val&

Experimental procedures as described in Example 10, except thatFmoc-Orn(Boc)-OH is replaced by Fmoc-D-Orn(Boc)-OH. The product wascharacterized by HPLC (t_(R) 9.89 min, Column A).

MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,478.06 [M+H]⁺1,500.15 [M+Na]⁺, 1,516.04 [M+K]⁺.

Example 12 H-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-(Z)Dhb-Val&

Starting with Fmoc-D-allo-Thr-D-allo-Ile-D-Val-Phe-ZDhb-O-TrtCl-resin(Step 2, Example 3), the Fmoc group was removed and after extensive DMFwashings, Boc₂O (546 mg, 5 equiv) and DIPEA (0.87 mL, 10 equiv) in DMFwere added and left to react for 2 h, when the ninhydrin test wasnegative. After DMF washings, Fmoc-Val-OH (848.2 mg, 2.5 mmol, 5 equiv)was coupled with DIPCDI (387 mg, 2.5 mmol, 5 equiv) in the presence ofDMAP (30.6 mg, 0.25 mmol, 0.5 equiv) and DIPEA (88 μL, 0.5 mmol, 1equiv) for 45 min. This coupling was repeated in the same conditionstwice. After removal of the Fmoc group as described in GeneralProcedures, the protected peptide was cleaved from the resin byTFA-CH₂Cl₂ (1:99) (5×30 sec). Filtrate was collected on H₂O (4 mL) andthe H₂O was partially removed under reduced pressure. ACN was then addedto dissolve the solid that appeared during the H₂O removal, and thesolution was lyophilized, to give 57 mg (75 μmol, 90% yield) of thetitle compound with a purity of >95% as checked by HPLC (Column A, t_(R)7.95 min).

ESMS, calcd for C₃₈H₆₀N₆O₁₀, 760.44. Found: m/z 762.3 [M+H]⁺.

Cyclization was carried out as in Step 6 of Example 1; and then the Bocwas removed with TFA-H₂O (19:1) (1 h). The solvent was removed underreduced pressure and dioxane was added (245 μL). The solvent was removedby evaporation under reduced pressure (the process was repeated threetimes), and then H₂O (1 mL) was added and lyophilized. The crude productwas purified by HPLC (Symmetry C₈ 5 μm, 30×100 mm), isocratic 30% ACN(+0.05% TFA) in water (+0.05% TFA), 20 mL/h, detection at 220 nm, togive the title product (50.2 mg, 67.6 μmol, 90% yield).

The product was characterized by HPLC (t_(R) 10.61 min, Column A) andfor MALDI-TOF-MS, calcd C₃₈H₅₈N₆O₉, 742.43. Found: m/z 743.40 [M+H]⁺,765.43 [M+Na]⁺, 781.43 [M+K]⁺.

Example 13(4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-Orn(BOC)-D-allo-Ile-OH

Experimental procedures as described in Example 4, except that thepeptide synthesis was initiated by incorporation of Fmoc-D-allo-Ile-OHto the Cl-TrtCl-resin. The product was characterized by HPLC (t_(R)10.25 min, Column A) and for MALDI-TOF-MS, Calcd C₅₁H₉₂N₈O₁₂, 1,008.68.Found: m/z 1,009.8.

Example 14(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D-allo-Ile-D-allo-Thr(&)-D-alto-Ile-D-Val-Phe-ZDhb-Val&

Experimental procedures as described in Examples 5 and 6, except that inExample 5 peptide from Example 1 is replaced by peptide from Example 12and peptide from Example 4 is replaced by peptide from Example 13. TheHPLC of the final product showed the presence of the epimeric peptide(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&(Example 15), which indicates racemization during the coupling stepbetween both protected peptides. The product was characterized by HPLC(t_(R) 7.92 min, Column A). MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93.Found: m/z 1,478.5 [M+H]⁺ 1,501.4 [M+Na]⁺, 1,517.6 [M+K]⁺.

Example 15(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-Ile-D-atlo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&

Experimental procedures as described in Examples 12-14, except thatFmoc-Ile-OH was used instead of Fmoc-D-allo-Ile-OH (Example 13).

(4S)-MeHex-D-Val-Thr(tBu)-Val-D-Val-D-Pro-Orn(Boc)-Ile-OH: (t_(R) 10.25min, Column A and MALDI-TOF-MS, Calcd C₅₁H₉₂N₈O₁₂, 1,008.68. Found: m/z1,009.5).

The final product was characterized by HPLC (t_(R) 8.02 min, Column A).MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,478.2 [M+H]⁺1,501.1 [M+Na]⁺, 1,517.3 [M+K]⁺.

Example 16(4S)-MeHex-D-Val-Thr-Val-D-Val-D-Pro-Orn-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-D-Val&

Experimental procedures as described in Examples 3,4 and 5 except thatFmoc-D-Val-OH was used instead of Fmoc-Val-OH (Example 3).

Alloc-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&:t_(R) 12.52 min, column A; MALDI-TOF-MS, calcd for C₅₃H₈₃N₉O₁₃,1,054.28. Found: m/z 1,056.6 [M+H]⁺.

-   -   -   -   H-Orn(Boc)-D-allo-Ile-D-allo-Thr(&)-D-allo-Ile-D-Val-Phe-ZDhb-Val&:                (t_(R) 9.23 min, Condition A and MALDI-TOF-MS, calcd                C₄₉H₇₉N₉O₁₁, 970.21. Found: m/z 972.4[M+H]⁺.

The final product was characterized by HPLC (t_(R) 9.23 min, Column A).MALDI-TOF-MS, calcd C₇₅H₁₂₄N₁₄O₁₆, 1,476.93. Found: m/z 1,479.6 [M+H]⁺1,502.5 [M+Na]⁺, 1,518.7 [M+K]⁺.

1. A process for the synthesis of kahalalide F or a mimic of kahalalideF comprising coupling a cyclic part with a side chain fragment.
 2. Aprocess according to claim 1, wherein the cyclic part already containsat least one of the side chain amino acids.
 3. A process according toclaim 1 or 2, wherein the compound obtained is kahalalide F with thefollowing formula:


4. A process according to claim 1 or 2, wherein the compound obtained isa mimic of kahalalide F which is structurally related to said compound,but differing in one or more of the following aspects: 1 to 7,especially 1 to 3, more especially 1 or 2, most especially 1, amino acidwhich is not the same as an amino acid of the parent compound; 1 to 10,especially 1 to 6, more especially 1 to 3, most especially 1 or 2,additional methylene groups in the side chain acyl group of the parentcompound; 1 to 10, especially 1 to 6, more especially 1 to 3, mostespecially 1 or 2, methylene groups omitted from the side chain acylgroup of the parent compound; 1 to 6, especially 1 to 3, more especially1 or 3, substituents added to or omitted from the side chain acyl groupof the parent compound; omission of the 5-methyl substituent from theacyl group of the side chain; and omission of the acyl group of the sidechain.
 5. A process according to claim 4, wherein the compound obtainedhas a 4(S)-methylhexanoyl group at the end of the side chain.
 6. Aprocess according to any of the preceding claims, wherein the couplingbetween the cyclic part and the side chain fragment is between D-Pro-9and L-Orn-8.
 7. A process according to any of claims 1 to 5, wherein thecoupling between the cyclic part and the side chain fragment is betweenL-Orn-8 and D-allo-Ile-7.
 8. A process according to any of claims 1 to5, wherein the coupling between the cyclic part and the side chainfragment is between D-Val-10 and D-Pro-9.
 9. A process according to anyof claims 1 to 5, wherein the coupling between the cyclic part and theside chain fragment is between D-allo-Ile-7 and D-allo-Thr-6.
 10. Aprocess according to any of claims 1 to 5, wherein the coupling betweenthe cyclic part and the side chain fragment is between L-Val-11 andD-Val-10.